Mixtures of volatile fatty acids having anti-fungal and anti-bacterial activity

ABSTRACT

Mixtures of at least two of propionic, butyric, or isobutyric acids are found to have greater fungicidal activity than any one of the acids alone on certain animal feedstuffs. The mixtures are also found to be anti-bacterial. The mixtures are useful in treating animal feedstuffs including grains, hay and silage, seed-meals, and high protein feedstuffs.

United States Patent [1 1 Herting et al.

[4 1 July 15, 1975 OTHER PUBLICATIONS R. Phytopathology 50, 781-784, (I960), Toxicity of Acetic Acid to Cladosporium Cucumerinum," by Strider et al.

Primary Examiner-Albert T. Meyers Assistant ExaminerD0ugIas W. Robinson Attorney, Agent, or FirmGeorge P. Chandler; Daniel B. Reece, III

[ 57] ABSTRACT Mixtures of at least two of propionic, butyric, or isobutyric acids are found to have greater fungicidal activity than any one of the acids alone on certain animal feedstuffs. The mixtures are also found to be antibacterial. The mixtures are useful in treating animal feedstuffs including grains, hay and silage, seed-meals, and high protein feedstuffs.

24 Claims, I Drawing Figure A 90% Propwmc 0% B 1 Pvopmmc 90% C 0% Proplomt: 90%

D 0% PvOpvomC 10% E 10% Propvomc 0'!- F I 90% Propmmc 0% G 74']. Pruvmmc 25% H 2/ Pmpiomc 79% I 0% Pwplomc 68% J 0% Propmruc I9!- K II A Propmmc 0% L I 76!- P'opmmc 0% M I 50% Pwpvomc 0% N I IOA mplomc 80% O l7% Pmmomc 17% P 40% PIODIOMC 0% Q I Prcpiomc 0% R Pmownic 10% PROPIONIC ACID Eutync 0 In Isobutyric Buly C O A lsohurync Bvvym: [0% lsohmyric Butync lsobut ric Bulyric 9O 56 Isobulyric Butyrtc IO lsobulyrl'c BuYyHC 0% lsonmync Bulync 0 A Ysatm m:

Butyn: 32 A Isohutyvic Butync 6| '1- Isobvlyric BuIync 89% Isobmyric Eulync 24% Isonulyn'c Butync 50% Isobulyric Bwyrr: 10% lsoburyvic Bulync 66 lsobutyvic Buiync 60 7G Isotlulync Burync 50% Isobu'yric Butym: 30% lsobulyric MIXTURES OF VOLATILE FATTY ACIDS HAVING ANTI-FUNGAL AND ANTI-BACTERIAL ACTIVITY This is a continuation-in-part of U.S. application Ser.

No. 262,932 filed Nov. 29, 1971, now abandoned.

This invention relates to mixtures of volatile fatty acids having greater anti-fungal activity than either acid alone as well as anti-bacterial activity and more particularly relates to mixtures of at least two of propionic, butyric, and isobutyric acids having the antifungal and anti-bacterial activity.

Deterioration and loss of various grain, oil seeds, and other feedstuffs is one of the most serious economic problems in the world today. The Food and Agricultural Organization, United Nations, estimated that 5% of all food grains harvested are lost before consumption. The World Health Organization has indicated the magnitude of the problem by quoting 20% post-harvest loss in Africa, Asia and Latin America, and 15% loss in the near East where spoilage in some areas affected 50% of the food in storage. Estimates of the amount lost due to deterioration resulting from fungi range from 0.5-l% of the world's production of grain. A solution to this problem of storage deterioration would affect significantly the world-wide shortage of food that presently exists.

Although these high losses are generally restricted to the less developed nations, which coincidentally are often in the temperate climates that favor fungal growth, significant economic losses also occur in the more developed nations such as the United States. Estimates of losses probably due to fungal damage in government or commercial warehouses range from about 0.5% of the soybeans to about 2% of the corn with a total value of $96,000,000.00. Farm storage losses are higher and range from 4.5% for wheat, oats, rye, barley and rice to 6.0% for corn and grain sorghum with a total value of $262,000,000.00. Though hay is somewhat less valuable than grains and oil seeds, losses are somewhat higher at 5% in commercial storage and 7% during farm storage.

Most of the damage to grain results from fungi which invade the grain after harvesting. The storage fungi comprise about a dozen species of Aspergillus, several species of Penicillium, a single species of Sporendonema and possibly a few species of yeast. Within the last decade, an increased awareness and understanding of a health hazard associated with the fungi has also come about. Under appropriate conditions, molds often produce mycotoxins, of which the best known one is Aflatoxin from Aspergillus Flavus. These toxins are of increasing concern because they are harmful to many domesticated animals, will produce hepatic cancer in at least several species, including rats and have been implicated with cirrhosis in children.

Key factors favoring the growth of mold are moderate temperatures and a high relative humidity. The length of time feedstuff is stored and the degree to which it is invaded by storage fungi are also important. Other factors such as presence of foreign material or fines and infestation by insects or mites may accelerate the process. For centuries man has attempted to minimize the influence of these factors with only minor success. Storage losses cited earlier demonstrate that even practices of more advanced agricultural societies have not achieved complete success.

The traditional approach in the United States has been to field dry crops as long as possible and then,

after harvesting, to dry further if necessary to the mois ture level presumed safe for storage. Some crops such as wheat or oats with shorter growing seasons and with earlier harvesting dates are more suitable for field drying than a crop such as corn which matures later. For any crop, however, these practices result in a relative flood of grain to be dealt with in a short span of time. When the weather is not cooperative, the grain is wetter than usual. Indeed, the farmer may be fortunate to be able to harvest the crop. While literally mountains of grain accumulate to await commercial drying, molding and heating can begin within 24 hours.

These problems are particularly acute with corn, the principal grain crop in the United States. In recent years the problems have been accentuated by the development of highly mechanized, efficient harvesting systems such as the picker-sheller. Although these systems increase the recovery of corn and spread the harvesting season over a longer period of time, they require at least 23% moisture in the grain for maximum effectiveness and thus compound the problem by producing more grain with high levels of moisture.

The basic way for preserving these grains has been drying, either by natural ventilation such as in crib stor age or by forced aeration, using either air at ambient temperature or air with supplemental heat in bin dryers, portable batch dryers, or continuous flow dryers. Artifical drying has permitted earlier harvest, better planning, long term storage and generally better quality.

When hot spots" develop or mold is observed during storage the grain may be re-dried. More frequently, however, the grain is blended out with sound grains or is turned either manually or by passing through an auger. The abrasion of such mechanical handling will break up hot spots and usually obscure the fact that mold has been growing but such measures are only temporary and do not stop mold growth.

The crop losses cited earlier testify that the present practices for preserving feedstuffs are not completely effective. Natural drying depends on the whims of weather. When crops are harvested wet, losses may be unavoidable simply because because dryers are either not available or their capacity is insufficient to handle the amount of grain and hay being dried. In other cases, the loss is due to carelessness in not drying sufficiently. Even drying of crops to moisture levels presumed safe for storage is no guarantee because diurnal temperature variations and other factors such as localization of fines may cause moisture migration within storage bins resulting in localized spots of ideal conditions for mold growth. Overdrying grain decreases the influence of the moisture factor for fungi but the resultant brittleness and cracking of the grain increase susceptibility to fungi, reduce grade, and decrease acceptibility by the food processor.

With grain, two other aspects of handling assume great importance and both are more or less encouraged by the official grain standards. The principal commercial classification of corn, for example, is grade 2 for which an allowable maximum of 15.5% moisture is permitted. Because the pricing of the corn is based on this moisture level, moisture is worth money and every effort is made to keep the moisture content as close as possible to this level without exceeding it. However, the maximum allowable moisture content for grains and feeds of the most common grade exceed by several percent the maximum moisture content that will permit safe storage for periods of time of a year or more. Since removal of this additional water is not only costly, but also results in shrinkage of weight without any compensatory increase in value, the grain processor tends to take his chances on storage losses.

A second practice in grain handling is co-mingling of grains. Grain that for any reason contains less than the allowable maximum moisture content can be mixed with grain containing higher than allowable moisture content so long as the average moisture content in the co-mingled grain meets the grain standards. Although this is legal, this practice permits a substrate that is more favorable to fungal growth than if every grain kernel were dried to the specifications of the grain standard.

Recently, there has been interest expressed in the use of formic, acetic, and propionic acids singly or in mixture for treating crops. This interest is exemplified by British Patents No. l,l49,3 14; NO. 1,155,485; No. 1,160,430; No. 1,219,857 and U.S. Pat. No. 3,595,665.

In accordance with our invention we have found that mixtures of at least two of propionic, butyric, and isobutyric acids when added to certain animal feedstuffs have a synergistic anti-fungal activity. This activity is found whether the mixtures are anhydrous mixture or a mixture diluted with water.

This anti-fungal activity has been observed when the mixture of at least two acids is -90% propionic, 0-90% butyric, or 0-90% isobutyric acids. These acid mixtures are also found to be effective when diluted with from about l090% by volume of the dilution water and in some instances the dilution with water further enhances the activity of said acid mixture.

The active mixtures found to be effective are further illustrated in FIG. 1 which is a three-phase trangular diagram. This trangular diagram is believed to be selfexplanatory especially when taken with the various points identified as A, B, C, etc., as listed in FIG. 1. The percentages are by volume of the composition.

According to one aspect of the invention, mixtures having the desired activity are of at least two of propionic, butyric, and isobutyric acids whose composition lies within the area on the triangular three-phase diagram of FIG. 1 defined by line A, G, M, H, B; line B, C; line C, 1,], D; line D, E; line B, K, P, Q, L, F; line F, A. According to another aspect of the invention the mixture of acids lies within the area on the triangular tree-phase diagram of FIG. 1 defined by line G, M, H; line H, N, line N, I; line I, J; line .I, K; line K, P, Q, L; line L, G. According to a still further aspect of the invention the mixture of acids lies within the area on the triangular three-phase diagram of FIG. 1 defined by line R, M; line M, N; line N, 0; line 0, P', line P, Q; line Q. R.

The mixtures may be diluted with from about to about 90% by volume of the dilution water, preferably about 40-90% and especially from about 40-60% by volume of the dilution water. Other preferred mixtures have been found to be 37.5% by volume of propionic acid, 25% by volume butyric acid, 37.5% isobutyric acid; 60% by volume propionic acid, 10% by volume butyric acid, and 30% by volume isobutyric acid; 40% by volume propionic acid, 60% by volume isobutyric acid; 50% by volume propionic acid, 50% by volume isobutyric acid; 10% by volume propionic acid, 90% by volume isobutyric acid. Other preferred dilutions have been found to comprise: 50% by volume of the acid mixture propionic acid, 25% by volume of the acid mixture butyric acid, 25% by volume of the acid mixture isobutyric acid diluted with 50% by volume of the dilu tion water; 50% by volume of the acids propionic acid, 25% by volume of the acids butyric acid, 25% by volume of the acid isobutyric acid diluted with by volume of the dilution water; 50% by volume of the acids propionic acid, and 50% by volume of the acid isobutyric acid, diluted with 90% by volume of the dilution water; 50% by volume of the acids propionic acid, 50% by volume of the acids isobutyric acid, diluted with 50% by volume of the dilution water; 60% by volume of the acids isobutyric acid, 40% by volume of the acids propionic acid, diluted with 50% by volume of the dilution water.

These acid mixtures and dilutions of acid mixtures may be applied by spraying the feedstuff as it enters an auger or other conveying system being used to load it into a storage area or by other techniques. Hay can be sprayed in the baling chamber, silage can be sprayed as it is chopped or as it is stored, or storage areas may be sprayed. Other application methods familiar to those skilled in the art may be used. The amount of volatile fatty acid mixture to be added depends on a number of factors including the type of feedstuff being treated, the amount of moisture in the feedstuff, the acid mixture used, and whether the acid mixture is anhydrous or diluted with water. We have found the amount of volatile fatty acid mixtures in a water dilution which must be added to have anti-fungal activity to vary from 0.17 grams per grams of corn having a moisture content of 20% to 0.69 grams per 100 grams of corn having a moisture content of 28.4%. Anhydrous mixtures of propionic, butyric and isobutyric acid have been found to have effective anti-fungal activity on 20% moisture content corn at levels of greater than 0.33 grams per 100 grams of corn; on 20% moisture content wheat the effective level has been found to be 0.41 gram or greater; on oats 0.51 gram or greater; on soybeans 0.42 gram or greater; on cottonseed meal 0.97 gram or greater; on soybean meal 048 gram per 100 grams or greater and on peanuts in the shell 0.68 gram per 100 grams or greater.

An aqueous dilution (75% water by volume of the dilutiori) of a mixture of 37.5% propionic, 25% butyric, and 37.5% isobutyric acids has been found to have antibacterial activity at a level of 1.0 gram of acid per 100 grams of tankage. Animal feedstuffs on which application of the binary and ternary acid mixtures according to one aspect of this invention have a synergistic fungicidal effect are observed to include corn, wheat, soybeans and peanuts in shell. Ternary acid mixtures according to another aspect of this invention are observed to have a synergistic effect on grain sorghum, hay, forage silage, and soybean meal. Certain binary mixtures of propionic and isobutyric acids are observed to have a synergistic fungicidal effect on corn and forage silages. Of course, other crops and animal feedstuffs can also be effectively treated with a fungicidal acid mixture disclosed herein. However, the synergistic effect obtained on the above mentioned feedstuffs has not been consistently observed on every feedstuff.

The following examples are further illustrative of the volatile fatty acid mixtures according to applicants invention. All percentages are percentages by volume unless otherwise noted. The effective fungicidal level (EFL) is weight given in grams/I grams, and is the lowest level of addition which inhibits mold growth. EFL is determined for corn and small grains in the following manner utilizing what is characterized as a Miniature Elevator test.

Polyethylene wash bottles (1,000 ml. capacity) are filled with a predetermined amount of a test material and distilled water added to adjust the moisture content to the desired level. The bottles are capped, shaken, rotated and placed on their sides in a cold room at 4C. for two days to equilibrate. During the equilibration period the bottles (hereinafter referred to as elevators) are shaken and turned (still on their sides) three times daily. A representative composite sample of a few grains from each elevator in a given series is tested for moisture content before the test to determine the effective fungicidal level (EFL) is started. The tests are run in series of 12 elevators each plus a control elevator to which no acid mixture is added. A predetermined amount of an acid mixture is added to the surface of the material to be tested in each test elevator. The amount added is varied until EFL is determined as described hereinafter.

After adding the acid mixture each elevator is immediately capped to prevent the escape of acid vapors, and is shaken and rotated vigorously. The caps are replaced by Bunsen valves and each series is set up in a warm room at 30C. The dispenser tube on each elevator, including the control, is connected to an air supply which is bubbled through distilled water at a rate of 0.3 liter per hour. The positive pressure insures an adequate air supply for maintaining the aerbic fungi in a high relative humidity atmosphere.

The incubation period for the miniature elevator" test is seven days, after which the contents of the elevator are examined grossly and with a dissecting micro scope. Duplicate samplings (about 20-30 kernels) of each grain from top", middle", and bottom of each elevator are plated on l0% salt-malt agar (Difco 0024 with sodium chloride added) and incubated 48 hours at 30C. and under greater than 70% relative humidity. The remaining contents of each elevator are then thoroughly mixed in a 6-inch by 9-inch porcelain pan. Twenty kernels are selected at random, placed in ml. distilled water in a 125 ml. Erlenmeyer flask, closed with a cotton plug and shaken four hours at room temperature (C.). Spore counts are done on a few drops of water from each flask by using a hemacytometer to count spores in the four corners and the middle square, as for a blood count. This method does not differentiate between living and dead spores and serves only as a guide to aliquot sizes to be taken from the distilled water for dilution in sterile water and subsequent plating on 10% salt-malt agar as modified from Buchanan and Gattani (E. D. Buchanan and R. E. Buchanan, Bacteriology". McMullen Co., New York (1936) p. 160 and M. L. Gattani,Phytopathology44. pp. 1 13-] 15 (1954). These plates and the Erlenmeyer flask containing the grains with water are incubated 48 hours as are plates containing untreated samples of the material tested. After 48 hours the following steps are carried out:

1. The plated kernels kernesl are examined grossly for any mold growth.

2. The plated spore dilutions are examined grossly for any mold growth.

3. The erlenmeyer flasks containing the material tested plus water are swirled and shaken and samples of water taken and placed on microscope slides. These water drops are examined microscopically. Five-hundred spores are counted with highpower (X 430) and percent present of those germinating determined.

This test is important as it not only reveals spore germination but also yeast and bacterial inhabitation and- /or growth. The plated kernels and plated spore solutions can be considered next in importance. Both show at a glance how well internal inoculum has been controlled. The gross examination of elevators shows only how well the external inoculum has been controlled. For example, an elevator can appear to be mold free by gross examination but this does not mean there has been a fungicidal action on the part of the anti-fungal until this finding is confirmed by a plating in the spore germination test. From an evaluation of these tests, the level of acid mixture at which inhibition of storage fungi (97-100%) occurs is determined and recorded. If inhibition does not occur in the range tested, higher levels of acid mixtures are tested until inhibition does occur. The lowest level at which inhibition occurs is considered to be the effective fungicidal level (EFL).

The evaluation of fungicidal activity (EFL) of volatile fatty acids (VFA) on soybean meal is performed as follows. Samples of 100 g. of soybean meal are placed in plastic bags (21 X 35 cm.). The VFA is added by syringe over the surface of the meal in each bag. The bags are closed, and the VFA are mixed into the meal by shaking, rotating and pressing the bag. After mixing, the meal is transferred to cloth mailing bags 15 X 24 cm.) with drawstring closures and incubated 7 days at 30C., 77% relative humidity. Untreated samples are included as controls. Microbiological examination of random samples of the incubated meal is carried out as described for corn and small grains, including plating of the meal and of spore solutions on l0% salt-malt agar and microscopic examination of the spore solutions.

The evaluation of volatile fatty acids for fungicidal activity (EFL) on peanuts is done in the same manner as was described for corn and small grains except the peanuts are placed in large beakers (100 g. peanuts/600 ml. beakers). VFA are spread on the walls of the beaker with a syringe. The beaker is covered immediately with 6 mil polyethylene sheeting, rotated and shaken, and incubated for 2 weeks at 30C., 77% relative humidity. Untreated samples are included as controls. After incubation, each beaker of peanuts is shelled. Each of two S-gram random samples of nutmeats and one 2-gram random sample of shells is plated on 10% salt-malt agar. The nuts and shells of each sample are then mixed together in 100 ml. distilled water. The water is decanted, and both washings and plates are incubated for 48 hours at 30C., 77% relative humidity. Microbiological evaluations are done as described for corn and small grains including gross and microscopic examination of the plated shells, nutmeats and spore solutions.

The evaluation of fungicidal activity (EFL) of volatile fatty acids on freshly-cut corn and forage silage is carried out as described for corn and small grains with the following exceptions. The silage is weighed into polyethylene bags (6 mil), the appropriate amount of VFA is added by syringe and the contents of the bags 7 are mixed by shaking. The treated silage is packed into silos" (1000 ml. plastic wash bottles from which the top one-fourth has been cut off) and the surface of the silage is covered with 6 mil polyethylene film held in vidual contributions to obtain a total expected EFL. The expected EFL is then divided by the actual EFL observed and converted to percentage by moving the decimal point two places to the right. If the quotient place with an aluminum insert of the same diameter as obtained is greater than 100 when converted to a perthe silo. Untreated samples are included as controls. cent, then there is synergistic effect. As a specific ex- The silos are stored in darkness at 30C. for 2 weeks. ample of this method of determining synergistic effect, Microbiological evaluation of random samples of the please refer to Example 1, Table l. Multiplying the persilage is carried out as described for grains including centage of propionic contained in the mixture plating of the silage and the spore solutions on 10% 10 times the effective fungicidal level of propionic when salt-malt agar as well as microscopic examination of the used alone (0.83) and repeating for butyric and isobuspore solutions. tyric acids, then adding these figures together and The evaluation of fungicidal activity (EFL) of volarounding two significant figures, one obtains an extile fatty acids on loose hay is carried out in the followpected EFL of 0.72. Dividing this expected EFL by the ing manner. One hundred gram samples of hay (recon- EFL observed of 0.34 a Synergistic Effect of 212% is stituted to 30% moisture content) are placed in polyobtained. ethylene bags (6 mil), the VFA is added by syringe, and In all tables following VFA stands for volatile fatty the contents of the bag are shaken to mix. Samples acids. from each bag are placed in open petri dishes (9 cm. dia.) and incubated 7 days at 30C., 77% relative hu- E I l th h 90 midity. Untreated samples are included as controls. Mi- Xamp es mug crobiological evaluation is performed as for the corn Mixtures of volatile fatty acids in the various volume and small grain samples. In brief, these procedures inrelationships given in Table l are tested to determine clude gross and microscopic examination of random their EFL as described above. Examples 46 through 54, samples of plated hay, of spore solutions and of plated 61 through 69, 71 through 73, and 75, 76, 78, 79, 8|, spore solutions. 82 and 84 through 86 of single acids are included for Synergistic Effect is determined in the following comparison purposes. It will be noted that the effect of manner. The expected EFL for various mixtures is dea mixture of two acids or three acids 18 greater than the termined by multiplying the percentage of each compocombined effect of any of the acids alone indicating the nent contained in the mixture times its individual EFL existence of synergistic action by the mixture. The rewhen used alone and then adding these expected indisults of the tests are shown in Table I.

Table I Antifungal Activity of Anhydrous Volatile Fatty Acid Mixtures Volatile Fatt Acid Moisture lso- Synergistic Ex- Material Content Prop. Butyric butyric EFL Effect ample Treated g./ 100 g.

1 Corn 20 10 80 I0 0.34 2l2 2 Corn 20 66 l7 l7 0.43 181 3 Corn 20 33.3 33.3 33.3 0.34 2l3 4 Corn 20 25 50 25 0.34 2] 3 5 Com 20 30 40 30 0.25 292 6 Corn 20 S0 25 25 0.34 222 7 Corn 20 ll l l 78 0.50 l32 8 Corn 20 80 l0 l0 0.52 I54 9 Corn 20 25 25 50 0.42 168 l0 Corn 20 l7 I7 66 0.34 200 ll Corn 20 30 I5 55 0.34 207 l2 Corn 20 60 l0 30 0.34 223 I3 Corn 20 55 I0 0.43 I79 l4 Corn 20 30 60 I0 0.42 177 15 Corn 20 20 70 I0 034 216 16 Corn 20 I0 70 20 0.34 210 l 7 Corn 20 I0 55 35 0.42 208 l8 Corn 20 I0 50 0.50 I37 l9 Corn 20 20 35 0.42 I69 20 Corn 20 35 25 40 0.34 212 2] Corn 20 40 45 I5 0.5l I47 22 Corn 2O I5 35 0.42 l7) 23 Wheat 20 37.5 25 37.5 0.44 l34 24 Soybeans 20 50 50 0.5 I 162 25 Soybeans 20 37.5 25 37.5 0.42 I93 26 Soybean 9 37.5 25 37.5 0.48 144 Meal 27 Peanuts 50 50 0.68 l 28 in shell 28 Peanuts 37.5 25 37.5 0.68 [28 in Shell 29 Corn 20 37.5 25 37.5 0.30 243 30 Corn 20 I0 90 0.50 l 28 3 I Corn 20 h 45 0.40 I67 32 Corn 20 90 I 0 0.58 123 33 Corn 20 it) 90 0.46 141 34 Corn 20 40 0.30 236 35 Corn 20 60 40 0.34 220 36 Corn 20 l0 0.6l I33 37 Corn 20 I0 90 0.56 I 47 38 Cnrn 20 50 50 0.35 22] 39 Corn 20 90 l0 0.63 [30 40 Corn 20 I) RI 0.45 I44 TABLE l-Continued Antifungal Activity of Anhydrous Volatile Fatty Acid Mixtures Volatile Fatty Acid Moisture lso- Synergistic Ex- Material Content Prop. Butyric butyric EFL Effect amp Treated g./l g.

4| Corn 68 32 0.45 154 42 Corn 20 l I 89 0.45 145 43 Corn 2O 76 24 0.45 174 44 Corn 20 21 79 0.45 165 45 Corn 20 74 26 0.45 178 46 Corn 20 100 0.83 47 Corn 20 100 0.72 48 Corn 20 100 0.63 49 Wheat 20 100 0.68 50 Wheat 20 100 0.58 51 Wheat 20 100 0.50 52 Soybeans 20 I00 0.89 53 Soybeans 20 I00 0.77 54 Soybeans 20 100 0.76 55 Corn 20 S0 50 0.41 165 56 Corn 20 50 50 0.33 221 57 Corn 20 25 75 0.42 162 58 Wheat 20 50 50 0.41 132 59 Wheat 20 50 50 0.42 140 60 Wheat 20 5O 50 0.43 146 61 Peanuts 100 0.89

in Shell 62 Peanuts I00 0.86

in Shell 63 Peanuts 100 0.85

in Shell 64 Soy bean 9 100 0.70

Meal 65 Soybean 9 100 0.86

Meal 66 Soybean 9 100 0.57

Meal 67 Hay 30 100 0.60 68 Hay 30 100 0.86 69 Hay 30 l00 1.04 70 Hay 3O 37 .5 25 37.5 0.48 173 71 Forage 80 100 0.74

Silage 72 Forage 80 100 2.88

Silage 73 Forage 80 100 0.71

Silage 74 80 37.5 25 37.5 0.48 263 Silage 75 Corn 65 100 1.00

Silage 76 Corn 65 100 1.04

Silage 77 Corn 65 10 90 0.80 130 Silage 78 Corn 55 100 1.24

Silage 79 Corn 55 100 1.04

Silage 80 Corn 55 10 90 0.86 123 Silage 81 Forage 77 I00 0.78

Silage 82 Forage 77 100 0.72

Silage 83 Forage 77 10 90 0.62 1 l8 Silage 84 Grain 20 100 0.73

Sorghum (Milo) 85 Grain 20 100 0 55 Sorghum (Milo) 86 Grain 20 100 0.48

Sorghum (Milo) 87 Grain 20 70 l5 l5 0 54 123 Sorghum (Milo) 88 Grain 20 15 70 15 0.38 147 Sorghum (Milo) 89 Grain 20 15 I 5 70 0.43 123 Sorghum (Milo) 90 Grain 20 37.5 25 37.5 0.50 118 Sor hum Examples 91 through 155 The effective fungicidal level of aqueous dilutions of volatile fatty acid mixtures is determined in accordance with the miniature elevator procedure outlined above. The results of this determination are shown in Table 11. Examples 137 through 139, 146 through 148, and 152 through 154 are presented for comparison purposes to show the effect of aqueous dilution of single volatile (Examples 46-48).

Table 11 Moisture Antifungal Activity of Aqueous Dilutions of Volatile Fatty Acid Mixtures Volatile Fatty Acids lso- Ratio Total Water Synergistic Ex- Material Content Prop. Buzric butvric Water VFA Mixture EFL Efi t ample Treated 7r VFA Added g./100 g. gJIOO g 9| Corn Z0 30 40 30 10:90 0 34 0.31 234 92 Corn 30 20:80 0.52 0.42 173 93 Corn 20 30 40 30 30:70 0.52 0.36 202 94 Com 20 30 40 30 40:60 0.61 0.37 197 95 Corn 20 30 40 30 :50 0.70 0.35 208 96 Corn 20 30 40 30 :40 0.87 0.35 208 97 Corn 20 30 40 30 :30 1.05 0.32 227 98 Corn 20 30 40 30 :20 1.14 0.23 316 99 Corn 18.4 30 40 30 10 1.94 0.17 427 100 Corn 18.4 30 40 30 90: 10 1.98 0.21 346 101 Corn 18.9 30 40 30 85115 1.44 0.21 346 102 Qom 20 50 25 25 10:90 0.34 0.31 243 103 Corn 20 50 25 25 20:80 0.43 0.34 221 104 Corn 20 50 25 25 30:70 0.52 0.36 209 105 Corn 20 50 25 25 40:60 0.61 0.36 209 106 Corn 20 5O 25 25 50:50 0.52 0.26 289 107 Corn 20 50 25 25 60:40 0.70 0.28 269 108 Com 20 50 25 25 70:30 0.61 0.18 417 109 Com 20 50 25 25 80:20 0.88 0.18 417 1 10 Corn 20 50 25 25 90:10 1.67 0.17 443 1 11 Corn 20 50 25 25 90: 10 1.50 0.15 502 112 Corn 20 30 40 30 90:10 1.27 0.14 518 I13 Com 18.5 33.3 33.3 33.3 90:10 2.01 0.21 340 114 Corn 18.5 80 10 10 90:10 2.06 0.26 307 115 Corn 18.5 10 80 10 85:15 2.09 0.29 249 116 Corn 18.5 10 10 80 85:15 2.09 0.29 227 117 Com 18.5 60 20 20 90:10 2.01 0.21 267 1 18 Corn 18.5 20 60 20 90:10 2.01 0.21 344 1 [9 Com 18.5 20 20 60 90: 10 2.01 0.21 329 120 Corn 18.5 20 40 40 90: 10 2.01 0.21 336 121 Corn 18.5 40 20 40 90:10 2.01 0.21 347 122 Corn 18.5 40 40 20 85:15 2.10 0.30 249 123 Com 18.5 30 40 30 90: 10 1.97 0.17 426 124 Com 18.5 50 25 25 90: 10 1.97 0.17 444 125 Com 24.8 33.3 33.3 33.3 80:20 1.99 0.39 183 126 Com 24.8 80 10 10 75:25 2.08 0.48 166 127 Corn 24.8 10 80 10 80:20 1.99 0.39 185 128 Corn 24.8 10 10 80 85:15 1.91 0.31 212 129 Com 24.8 60 20 20 85:15 1.91 0.31 248 130 Corn 24.8 20 60 20 80:20 1.99 0.39 186 131 Com 24.8 20 20 60 80:20 2.02 0.42 163 132 Corn 24.8 20 40 40 80:20 2.02 0.42 168 133 Corn 24.8 40 20 40 80:20 1.99 0.39 186 134 Corn 24.8 40 40 20 80:20 1.95 0.35 213 135 Corn 24.8 32 15 53 80:20 1.94 0.34 208 136 Corn 185 35 20 45 90: 10 1.98 0.18 393 137 Com 20 100 50:50 0.90 0.45 138 Com 20 100 50:50 0.84 0.42 139 Corn 20 100 50:50 0.71 0.36 140 Com 20 50 50 50:50 0.59 0.30 225 141 Corn 20 50 50 i 50:50 0.51 0.25 292 142 Corn 20 24 76 50:50 0.51 0.25 272 143 Corn 248 50 50 80:20 1.99 0.39 199 144 Corn 24.8 50 50 85: 1 5 1.91 0.31 218 145 Corn 24.8 50 50 85: 1 5 1.91 0.31 236 I46 Corn 24.8 100 75:25 2.12 0.52 I47 Com 24.8 100 80:20 2.06 0.46 I48 Corn 24.8 100 80:20 1.99 0.39 14) Cum 18.5 50 50 90:10 2.05 0.25 310 I50 Corn 18.5 50 50 90:10 2.05 0.25 270 I51 Corn 185 50 50 90: 10 2.01 0.21 348 152 Corn 185 100 75:25 2.40 0.60 153 Corn 18.5 100 80:20 2.22 0.42 154 Corn 18.5 100 85:15 2.09 0.29 155 Com 20 S0 50 50:50 2.05 0.25 310 But unacceptable because of yeast growth Example 156 An aqueous mixture of volatile fatty acids is tested to determine the effective level required to inhibit or eradicate salmonella in tankage. Commercially prepared tankage is sterilized by autoclaving 15 minutes at psi. Salmonella gallinarum, a relatively innocuous salmonella species obtained in a selenite-cystine broth culture, is introduced (1 ml.) into 100 ml. of selenitecystine broth and incubated for 48 hours at 37C. A high bacterial count is thus obtained and added at a level of I00 ml. per I00 grams sterile tankage. After adding the culture to the tankage, enough sterile physiological saline solution is added to form a thick slurry". This mixture is stirred thoroughly and incubated for 24 hours at 37C. Additional tankage is then added with thorough mixing until the mixture is just moist. This artificially contaminated tankage is dried 2 hours at 37C. before use. Eight I00 gram portions of the dry contaminated tankage are weighed into separate 32-ounce clear screw-cap bottles. The bottles containing tankage are treated by adding various amounts ofa volatile fatty acid mixture of 37.5% propionic, 25% butyric, 37.5% isobutyric acids to the tankage in the bottles along with 3.75 ml. of water. Each bottle is capped and mixed on a rotary mixer for 30 minutes and the bottles incubated at 37C. with the caps placed loosely on top. After 24 hours, 48 hours, and 1 week, two ten-gram samples are removed from each bottle. One sample is added to selenite-cystine broth, the other sample is added to tetrathionate broth (Difco 0l04-0l grams tankage per I00 ml. broth) and both are incubated for 24 hours at 37C. After incubation, 3 mm. loopfuls of each broth culture are streaked on SS Agar (Salmonella-Shigella Agar, Difco 0074-01) and on BG Agar (Brilliant Green Agar, Difco 00285-01). The petri plates are incubated 24 hours at 37C. and examined carefully for the presence of salmonella colonies.

After one week, the mixture is found to be salmonel- Iacidal at one ml. acid mixture per 100 grams tankage but not salmonellacidal at 0.75 ml. acid mixture per 100 grams of tankage.

In accordance with applicants invention there has been produced a mixture of volatile fatty acids having a high degree of anti-fungal and anti-bacterial activity on a variety of crops and animal feed stuffs.

Although the invention has been described in considerable detail with reference to certain preferred embodiments thereof, it will be understood that variations and modifications can be effected without departing from the spirit and scope of the invention as described hereinabove.

We claim:

1. A synergistic fungicidal mixture for animal feedstutfs selected from the group consisting of corn, wheat, soybeans and peanuts in shell consisting essentially of at least two of propionic, butyric, and isobutyric acids whose composition lies within the area on the triangular three phase diagram of FIG. 1 defined by:

A. line A, G, M, H, B;

line B, C;

C. line C, I, .I, D;

D. line D, E;

E. line E, K, P, Q, L, F; and

F. line F, A.

2. The mixture of claim 1 wherein the composition lies within the area on the triangular three-phase diagram of FIG. I defined by:

A. line G, M, H;

B. line H, N;

C. line N, I;

D. line I, J;

E. line J, K;

F. line K, P, Q, L; and

G. line L, G.

3. The mixture of claim 1 wherein the composition lies within the area on the triangular three phase dia gram of FIG. 1 defined by:

A. line R, M;

8. line M, N;

C. line N, O,

D. line 0, P;

E. line P, Q; and

F. line Q, R.

4. An aqueous solution of the acid mixture of claim 1 which is from about 40-90% by volume water.

5. An aqueous solution of the acid mixture of claim 2 which is from about 4090% by volume water.

6. An aqueous solution of the acid mixture of claim 3 which is from about 4090% by volume water.

7. The mixture of claim 1 wherein said acid mixture is about 40% by volume propionic acid and about 60% by volume of the isobutyric acid.

8. An aqueous solution of the acid mixture of claim 7 which is about 50% by volume water.

9. A synergistic fungicidal mixture for animal feedstuffs selected from the group consisting of grain sorghum, hay, forage silage, and soybean meal consisting essentially of about 10 to by volume of the mixture of propionic acid, about l0 to 80% by volume of the mixture butyric acid and about l0 to 80% by volume of the mixture isobutyric acid.

10. An aqueous solution of the acid mixture of claim 9 which is from about 4090% by volume water.

11. The mixture of claim 9 wherein said acid mixture is about 37.5% by volume of the mixture propionic acid, 25% by volume of the mixture butyric acid and 37.5% by volume of the mixture isobutyric acid.

12. An aqueous solution of the acid mixture of claim 11 which is about 50% by volume water.

13. A synergistic fungicidal mixture for animal feedstuifs selected from the group consisting of corn, corn silage and forage silage consisting essentially of about l0% by volume of the mixture propionic acid and about by volume of the mixture isobutyric acid.

14. An aqueous solution of the acid mixture of claim 13 which is from about 40-90% by volume water.

15. A method for preventing the growth of mold in animal feedstuffs selected from the group consisting of corn, wheat, soybeans and peanuts in shell which comprises treating the feedstufi' with a mold inhibiting quantity of a mixture of at least two of propionic, butyric, and isobutyric acids according to claim 1.

16. A method for preventing the growth of mold in animal feedstuffs which comprises treating the feedstuff selected from the group consisting of corn, wheat, soybeans and peanuts in shell with a mold inhibiting quantity of a mixture of at least two of propionic, butyric, and isobutyric acids according to claim 4.

17. A method for preventing the growth of mold in animal feedstuffs which comprises treating the feedstuff selected from the group consisting of corn, wheat, soybeans and peanuts in shell with a mold inhibiting quantity of a mixture of propionic and isobutyric acids according to claim 7.

18. A method for preventing the growth of mold in animal feedstuffs selected from the group consisting of corn, wheat, soybeans and peanuts in shell which comprises treating the feedstuff with a mold inhibiting quantity of a mixture of propionic and isobutyric acids diluted with water according to claim 8.

l9. A method for preventing the growth of mold and bacteria in animal feedstuffs selected from the group consisting ofgrain sorghum, hay, forage silage and soy bean meal which comprises treating the feedstuff with a mold inhibiting quantity of a mixture of propionic, butyric and isobutyric acids according to claim 9.

20. A method for preventing the growth of mold in animal feedstuffs selected from the group consisting of grain sorghum, hay, forage silage and soybean meal which comprises treating the feedstuff with a mold inhibiting quantity of a mixture of propionic, butyric and isobutyric acids diluted with water according to claim 10.

21. A method for preventing the growth of mold in animal feedstuffs selected from the group consisting of grain sorghum, hay, forage silage and soybean meal which comprises treating the feedstuff with a mold inhibiting quantity of a mixture of propionic, butyric and isobutyric acids according to claim ll.

22. A method for preventing the growth of mold in animal feedstuffs selected from the group consisting of grain sorghum, hay, forage silage and soybean meal which comprises treating the feedstuff with a mold inhibiting quantity of a mixture of propionic, butyric and isobutyric acids diluted with water according to claim 12.

23. A method for preventing the growth of mold in animal feedstuff selected from the group consisting of corn, corn silage and forage silage which comprises treating the feedstuff with a mold inhibiting quantity of a mixture of propionic and isobutyric acids according to claim 13.

24. A method for preventing the growth of mold in animal feedstuffs selected from the group consisting of corn, corn silage and forage silage which comprises treating the feedstuff with a mold inhibiting quantity of a mixture of propionic and isobutyric acids diluted with water according to claim 14.

3,895,116 Dated July 15, 1975 Patent No.

Inventods) David C. Herting and Emma-Jane E. Drury It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

In the drawing, line "M" should read -M 50% Propionic 507" Butyric OZ Isobutyric- Signed and Scaled this twenty-fifth Day Of May 1976 [SEAL] Arrest.

RUTH C. MASON C. MARSHALL DANN Arresting Officer (ummissinner oj'lalems and Trademarks 

1. A SYNERGISTIC FUNGICIDAL MIXTURE FOR ANIMAL FEEDSTUFFS SELECTED FROM THE GROUP CONSISTING OF CORN, WHEAT, SOYBEANS AND PEANUTS IN SHELL CONSISTING ESSENTIALLY OF AT LEAST TWO OF PROPIONIC, BUTYRIC, AND ISOBUTYRIC ACIDS WHOSE COMPOSITION LIES WITHIN THE AREA ON THE TRIANGULAR THREE PHASE DIAGRAM OF FIG. 1 DEFINED BY: A. LINE A. G, M, H, B, LIME B, C, C. LINE C, I, J, D, D, LINE D, E, E. LINE E, K, P, Q, L, F, AND F. LINE F, A.
 2. The mixture of claim 1 wherein the composition lies within the area on the triangular three-phase diagram of FIG. 1 defined by: A. line G, M, H; B. line H, N; C. line N, I; D. line I, J; E. line J, K; F. line K, P, Q, L; and G. line L, G.
 3. The mixture of claim 1 wherein the composition lies within the area on the triangular three phase diagram of FIG. 1 defined by: A. line R, M; B. line M, N; C. line N, O; D. line O, P; E. line P, Q; and F. line Q, R.
 4. An aqueous solution of the acid mixture of claim 1 which is from about 40-90% by volume water.
 5. An aqueous solution of the acid mixture of claim 2 which is from about 40-90% by volume water.
 6. An aqueous solution of the acid mixture of claim 3 which is from about 40-90% by volume water.
 7. The mixture of claim 1 wherein said acid mixture is about 40% by volume propionic acid and about 60% by volume of the isobutyric acid.
 8. An aqueous solution of the acid mixture of claim 7 which is about 50% by volume water.
 9. A synergistic fungicidal mixture for animal feedstuffs selected from the group consisting of grain sorghum, hay, forage silage, and soybean meal consisting essentially of about 10 to 80% by volume of the mixture of propionic acid, about 10 to 80% by volume of the mixture butyric acid and about 10 to 80% by volume of the mixture isobutyric acid.
 10. An aqueous solution of the acid mixture of claim 9 which is from about 40-90% by volume water.
 11. The mixture of claim 9 wherein said acid mixture is about 37.5% by volume of the mixture propionic acid, 25% by volume of the mixture butyric acid and 37.5% by volume of the mixture isobutyric acid.
 12. An aqueous solution of the acid mixture of claim 11 which is about 50% by volume water.
 13. A synergistic fungicidal mixture for animal feedstuffs selected from the group consisting of corn, corn silage and forage silage consisting essentially of about 10% by volume of the mixture propionic acid and about 90% by volume of the mixture isobutyric acid.
 14. An aqueous solution of the acid mixture of claim 13 which is from about 40-90% by volume water.
 15. A method for preventing the growth of mold in animal feedstuffs selected from the group consisting of corn, wheat, soybeans and peanuts in shell which comprises treating the feedstuff with a mold inhibiting quantity of a mixture of at least two of propionic, butyric, and isobutyric acids according to claim
 1. 16. A method for preventing the growth of mold in animal feedstuffs which comprises treating the feedstuff selected from the group consisting of corn, wheat, soybeans and peanuts in shell with a mold inhibiting quantity of a mixture of at least two of propionic, butyric, and isobutyric acids according to claim
 4. 17. A method for preventing the growth of mold in animal feedstuffs which comprises treating the feedstuff selected from the group consisting of corn, wheat, soybeans and peanuts in shell with a mold inhibiting quantity of a mixture of propionic and isobutyric acids according to claim
 7. 18. A method for preventing the growth of mold in animal feedstuffs selected from the group consisting of corn, wheat, soybeans and peanuts in shell which comprises treating the feedstuff with a mold inhibiting quantity of a mixture of propionic and isobutyric acids diluted with water according to claim
 8. 19. A method for preventing the growth of mold and bacteria in animal feedstuffs selected from the group consisting of grain sorghum, hay, forage silage and soybean meal which comprisEs treating the feedstuff with a mold inhibiting quantity of a mixture of propionic, butyric and isobutyric acids according to claim
 9. 20. A method for preventing the growth of mold in animal feedstuffs selected from the group consisting of grain sorghum, hay, forage silage and soybean meal which comprises treating the feedstuff with a mold inhibiting quantity of a mixture of propionic, butyric and isobutyric acids diluted with water according to claim
 10. 21. A method for preventing the growth of mold in animal feedstuffs selected from the group consisting of grain sorghum, hay, forage silage and soybean meal which comprises treating the feedstuff with a mold inhibiting quantity of a mixture of propionic, butyric and isobutyric acids according to claim
 11. 22. A method for preventing the growth of mold in animal feedstuffs selected from the group consisting of grain sorghum, hay, forage silage and soybean meal which comprises treating the feedstuff with a mold inhibiting quantity of a mixture of propionic, butyric and isobutyric acids diluted with water according to claim
 12. 23. A method for preventing the growth of mold in animal feedstuff selected from the group consisting of corn, corn silage and forage silage which comprises treating the feedstuff with a mold inhibiting quantity of a mixture of propionic and isobutyric acids according to claim
 13. 24. A method for preventing the growth of mold in animal feedstuffs selected from the group consisting of corn, corn silage and forage silage which comprises treating the feedstuff with a mold inhibiting quantity of a mixture of propionic and isobutyric acids diluted with water according to claim
 14. 